GAI MoRFs Regulate Cleft and Channel Binding Pathways for Gibberellin in GID1A

Abstract The hormone gibberellin (GA) promotes arabidopsis growth by enhancing binding between GA Insensitive DELLA transcriptional repressors and GA Insensitive Dwarf 1 (GID1) receptors to regulate DELLA degradation. The binding mechanism for GA was elucidated by employing a computational study of dissociations of the N-terminus of the DELLA family member GAI (GA Insensitive transcriptional repressor) from the GID1A receptor in the presence and absence of bound GA, and of GA from GID1A in the presence and absence of GAI. The tRAMD method was employed to deduce egression pathways for a diverse set of GA molecules (GA (x) ). Two pathways in the form of a newly identified cleft and a previously identified channel are prevalent. The cleft pathway is open in the absence of GAI. Upon GAI binding, the cleft route is blocked, resulting in a slower process for GA (x) to exit and enter the binding pocket through the channel. Several binding pocket residues are identified as gate-keepers to the channel. Molecular recognition features (MoRFs) found in the disordered signaling protein GAI affect GA (x) binding and GID1A dynamics. A three-step synergistic binding cycle is proposed where GAI MoRFs regulate the process. Rapid binding takes place through the cleft where little to no distinctions are made between major and less active forms of GA (x) . After GAI is bound to the GA (x) · GID1A complex, the channel supports a rectification process that increases the retention of major active forms of GA within the binding pocket. Both the cleft and channel contact residues to GA (x) are markedly conserved in a GID1 phylogeny, suggesting this binding process in the GID1 · DELLA GA-receptor complex represents a general paradigm for GA binding. Non-specific GA binding assists binding of GAI, which then helps to select the major active forms of the hormone and induce a downstream signalling cascade in response to bioactive GA. Non-expert Summary Statement Gibberellins are plant hormones essential for growth and development. The DELLA proteins are a disordered family of repressors that transcriptionally repress GA responsive genes. Degradation of DELLA proteins in response to GA results in GA-responsive genes being upregulated. Binding of GA to the GA-Insensitive Dwarf 1 receptor (GID1) facilitates binding of DELLA to the GA · GID1 complex. Through computational modelling and phylogenetic analyses, we identified a new GA binding cleft that is blocked by DELLA binding and a three-step mechanism for the GA · DELLA · GID1 complex that also involves the known GA binding channel. We propose a dual (cleft/channel) pathway that allows access to the binding pocket as a paradigm for selection of specific GA forms among a mixture of major active and inactive forms. The cleft is less selective, but preference for active GA in the binding pocket of GID1A is amplified by expunging inactive GA forms, followed by recruiting active forms through the more selective channel. This mechanism allows plants to sense concentration changes of GA with high specificity to enable certain GA variants to trigger specific signalling events. These novel insights into the receptor mechanism in part may explain the large number of different GA forms that exist in nature. Graphical Abstract